Reverse transcriptases (RTs) are employed in biotechnology to synthesize cDNA copies of RNAs for a variety of applications, including RT-PCR and qRT-PCR, construction of cDNA libraries, generation of probes for microarrays, and conventional and next-generation RNA sequencing. The synthesis of cDNAs corresponding to long polyadenylated RNAs can be accomplished by using random hexamer primers or an oligo(dT)-containing primer, which is complementary to the poly(A) tail. However, the strand-specific cloning and sequencing of cDNAs corresponding to non-polyadenylated RNAs, such as miRNAs or protein-bound RNA fragments, typically requires ligating DNA, RNA or chimeric RNA/DNA oligonucleotide adaptors containing PCR-primer-binding sites to the termini of the RNA or cDNA strand (Lau et al. 2001; Levin et al. 2010; Lamm et al. 2011). The adaptors are commonly ligated to the RNA template using RNA ligases, either sequentially to the 3′ and 5′ ends of the RNA (e.g., Roch 454 Life Sciences® sequencing and Illumina® next-generation sequencing) or simultaneously to both RNA ends (e.g., SOLiD™ next-generation sequencing) (Linsen et al. 2009). For some applications, the first adaptor is ligated to the 3′ end of the RNA for reverse transcription and the second adaptor to the 3′ end of the resulting cDNA (e.g., cross-linking and analysis of cDNAs (CRAC) of protein-bound RNA fragments; Granneman et al. 2009). In one variation, the ligation of a second adaptor is circumvented by using a non-templated nucleotide addition reaction of the reverse transcriptase to add C-residues to the 3′ end of the cDNA, enabling annealing of a second adaptor containing complementary G-residues for second-strand synthesis (Zhu et al. 2001). In another variation, the ligation of a second adaptor is circumvented by circularization of the cDNA followed by linearization and PCR amplification using bidirectional primer binding sites in the first adaptor, for example in individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP, König et al. 2010) or genome-wide in vivo analysis of translation with nucleotide resolution using ribosome profiling (Ingolia et al. 2009).
Unfortunately, although the attachment of oligonucleotide adaptors is needed for facile PCR amplification for the cloning and sequencing of cDNAs corresponding to non-polyadenylated RNAs and RNA fragments, the use of ligases to attach adaptors is a time-consuming, expensive, and inefficient step. Moreover, RNA ligases commonly used for adaptor ligation have distinct nucleotide preferences for the ends being ligated, leading to biased representation of cDNAs in the constructed libraries (Linsen et al. 2009; Levin et al. 2010).
Retroelements, genetic elements that encode RTs, are divided into two major families denoted LTR-containing retroelements and non-LTR-containing retroelements (Xiong and Eickbush 1990). Retroviruses, whose RTs are commonly used in biotechnology, are well-known examples of LTR-containing retroelements. Non-LTR-retroelements are a diverse family of RT-encoding elements that includes retroplasmids, non-LTR-retrotransposons, retrons, and mobile group II introns (Xiong and Eickbush 1990). Mobile group II introns consist of a catalytically active intron RNA (“ribozyme”) and an intron-encoded RT, which function together to promote RNA splicing and intron mobility (Lambowitz and Zimmerly 2010). Group II intron RTs typically consist of four conserved domains: RT, which contains seven conserved sequence blocks (RT1-7) found in the fingers and palm regions of retroviral RTs; X, a region required for RNA splicing activity corresponding at least in part to the thumb domain of retroviral RTs; D, a DNA-binding domain involved in DNA target site recognition; and En, a DNA endonuclease domain that cleaves the DNA target site to generate the primer for reverse transcription (Blocker et al. 2005; Lambowitz and Zimmerly 2010). The En domain is missing in some group II intron RTs, which instead use nascent strands at DNA replication forks to prime reverse transcription (Zhong and Lambowitz 2003; Lambowitz and Zimmerly 2010). The RT and X/thumb domains of group II intron RTs are larger than those of retroviral RTs due to an N-terminal extension, an additional N-terminal conserved sequence block (RT-0), and insertions between the conserved sequence blocks in the RT and X/thumb domain (Lambowitz and Zimmerly 2010). RT-0 and some of the insertions between conserved sequence blocks in the RT domain are also found in other non-LTR-retroelement RTs (Blocker et al. 2005). Unlike retroviral RTs, group II intron and non-LTR-retroelement RTs lack an RNase H domain.
The RTs encoded by retroplasmids and non-LTR-retrotransposons have been found to differ from retroviral RTs in being able to template switch directly from an initial RNA template to the 3′ end of a new RNA template that has little or no complementarity to the 3′ end of the cDNA synthesized from the initial template (Chen and Lambowitz 1997; Bibillo and Eickbush 2002, 2004; Kennell et al. 1994).